Many plant species store triacylglycerols (TAGs) in their seeds as a carbon reserve. These TAGs are the major source of energy and carbon material that supports seedling development during the early stages of plant life. Vegetable oils from soybean (Glycine max), Brassica (Brassica napus or B. rapa), sunflower (Helianthus annuus) and many other oilseed crops are also an important source of oil for the human diet or industrial applications including, but not limited to biofuels, biolubricants, nylon precursors, and detergent feedstocks. The degree and/or amount of polyunsaturated fatty acids of vegetable oils are characteristic and determinative properties with respect to oil uses in food or non-food industries. More specifically, the characteristic properties and utilities of vegetable oils are largely determined by their fatty acyl compositions in TAG.
Major vegetable oils are comprised primarily of palmitic (16:0), stearic (18:0), oleic (18:1cis Δ9), linoleic (18:2cis Δ9, 12), and α-linolenic (18:3cis Δ9, 12, 15 or C18:3) acids. Palmitic and stearic acids are, respectively, 16 and 18 carbon-long, saturated fatty acids. Oleic, linoleic, and linolenic acids are 18-carbon-long, unsaturated fatty acids containing one, two, and three double bonds, respectively. Oleic acid is referred to as a monounsaturated fatty acid, while linoleic and linolenic acids are referred to as polyunsaturated fatty acids. Modifications of the fatty acid compositions have been sought after for at least a century in order to provide optimal oil products for human nutrition and chemical (e.g., oleochemical) uses (Gunstone, 1998, Prog Lipid Res 37:277; Broun et al., 1999, Annu Rev Nutr 19:107; Jaworski et al, 2003, Curr Opin Plant Biol 6:178). In particular, the polyunsaturated fatty acids (18:2 and 18:3) have received considerable attention because they are major factors that affect nutritional value and oil stability. However, while these two fatty acids provide essential nutrients for humans and animals, they increase oil instability because they comprise multiple double bonds that may be easily oxidized during processing and storage.
The desaturation of 18:1 into 18:2 is a critical step for synthesizing polyunsaturated fatty acids. During storage lipid biosynthesis, this reaction is known to be catalyzed by the fatty acid desaturase, FAD2, a membrane-bound enzyme located on the endoplasmic reticulum (ER) (Browse and Somerville, 1991, Annu Rev Plant Physiol Plant Mol Biol 42:467), which has delta-12 fatty acid desaturase activity. The FAD2 substrate 18:1 must be esterified on the sn-2 position of phosphatidylcholine (PC) (Miguel and Browse, 1992, J Biol Chem 267:1502; Okuley et al., 1994, Plant Cell 6:147), which is the major membrane phospholipid of plant cells. Not surprisingly, therefore, down-regulation of FAD2 (and FAD3) genes has become a preferred strategy for avoiding the need to hydrogenate vegetable oils and the concomitant production of undesirable trans fatty acids. For example, soybean has both seed-specific and constitutive FAD2 desaturases, so that gene silencing of the seed-specific isoform has allowed the production of high-oleate cultivars (>88% 18:1 in the oil) in which membrane unsaturation and plant performance are largely unaffected.
There are several reports on silencing of FAD2 genes in order to increase the levels of oleic acid. Stoutjesdijk et al., 2000 (Biotech Soc Trans 28:938) discloses B. napus plants carrying a Δ12-desaturase (FAD2) co-suppression contstruct having oleic acid levels of up to 89%. Chen et al., 2006 (J Plant Physiol Mol Biol 32: 665) report seed-specific FAD2 gene silencing in Brassica napus, which results in oleic acid content in transgenic plant seeds of 83.9%. They further report that the transgenic plants with high oleic acid grow normally and without disadvantageous agronomic traits. Peng et al., 2010, Plant Cell Rep 29:317 disclose Brassica napus plants in which FAD2 and the fatty acid elongase 1 (FAE1) genes are simultaneously silenced, reaching oleic acid levels of up to 85%. WO1994/011516 report gene silencing of FAD2 genes in Brassica napus resulting in levels of oleic acid of up to 85%. WO2013/112523.
There are also several mutant Brassica plants described with increased levels of oleic acid: WO97/21340 and WO98/56239 disclose Brassica lines with increased levels of oleic acid, comprising amino acid substitutions in the FAD2 proteins; WO2006/079567 describes a high oleic Brassica napus line comprising a nucleotide deletion in a FAD2 gene, leading to a premature translation stop, whereas WO2013/049356 also describes a high oleic Brassica napus line comprising a premature translation stop codon in the FAD2 gene leading to a truncated protein; WO2007138444, WO2007/099459, WO2007/107590 and WO2008/084107 describe several mutations in FAD2 genes in Brassica lines with high levels of oleic acid.
Wells et al., 2014 (Mol Breeding 33: 349) and WO2012/117256 describe oilseed rape cultivars with a lower than usual polyunsaturated fatty acids content, which has non-functional alleles at three of the four orthologous FAD2 loci. Further mutations in the remaining functional FAD2 copy, leading to amino acid substitutions or premature stop codons, result in a polyunsaturated fatty acids content of about 6%, and an oleic acid content of about 84%.
Significantly, however, canola and other oilseed plants have only constitutive FAD2 enzymes. Therefore, in canola and other such constitutive FAD2 crops, silencing or down-regulation of FAD2 not only alters the fatty acid composition of the storage triacylglycerol (TAG) in seeds, but also of the cellular membranes. For example, the defective FAD2 in the Arabidopsis mutant fad2 alters fatty acid compositions of seeds as well as vegetable tissues, and severely compromises plant growth (Browse and Somerville, supra). FAD2 mutations and silencing that produce the highest 18:1 levels in the oil also reduce membrane unsaturation in vegetative and seed tissues, resulting in plants that germinate and grow poorly. As a result, only partial downregulation of FAD2 expression is possible, producing approximately 70-75% 18:1 in the oil of commercial cultivars such as Nexera/Natreon (Dow AgroSciences) and Clear Valley 75 (Cargill).
The object of the current invention is to provide Brassica FAD2 alleles for the production of plants with high levels of oleic acids while maintaining normal agronomic development and, optionally, to combine the FAD2 alleles with FADS alleles to produce plants with high levels of oleic acids and low levels of linolenic acids.